Microfluidic Engineered Gelatin Methacryloyl Microsphere Scaffolds Loaded with Extracellular Vesicles Derived from Mesenchymal Stromal Cells Promote Functional Recovery in Rats with Spinal Cord Injury

Spinal cord injury (SCI) leads to significant locomotor, sensory, and autonomic deficits and remains refractory to current therapies. Extracellular vesicles from mesenchymal stromal cells (MSC-EVs) are potential acellular therapeutic strategy for tissue regeneration, but rapid clearance and poor lesion retention limit their efficacy; combining MSC-EVs with biomaterial depots can improve local retention and sustained release. Here, we engineered porous gelatin methacryloyl (GelMA) microspheres using a self-designed simple microfluidic device. MSC-EVs were physically loaded into porous GelMA microspheres, which were subsequently embedded into a GelMA scaffold to form GelMA microsphere-embedded GelMA scaffolds loaded with MSC-EVs (GMS-EVs scaffolds). Based on the satisfactory elastic modulus, suitable porous organized structure, and good biocompatibility, the GMS-EVs scaffolds could achieve local retention and sustained release of MSC-EVs both in vivoand in vitro. Implantation of GMS-EVs scaffolds showed significantly improved motor recovery, reduced glial scar formation, and enhanced neuronal regeneration compared to both SCI and GelMA-only controls in a rat spinal cord hemisection model. Mechanistically, these beneficial effects of GMS-EVs scaffolds were associated with the stimulation of the phosphoinositide 3-kinase/protein kinase B (PI3K-AKT) and extracellular signal-regulated kinase (ERK) signaling pathways in SCI rats. Overall, this study highlights the potential of scaffolds based on porous GelMA microspheres combined with MSC-EVs to enhance tissue repair in the injured spinal cord environment.